Anti-Inflammatory Effect of Xanthones from Hypericum beanii on Macrophage RAW 264.7 Cells through Reduced NO Production and TNF-α, IL-1β, IL-6, and COX-2 Expression

Hypericum beanii N. Robson, a perennial upright herb, predominantly inhabits temperate regions. This species has been utilized for the treatment of various inflammation-related diseases. One new xanthone 3,7-dihydroxy-1,6-dimethoxyxanthone (1) and twenty-three known xanthones (2–24) were isolated from the aerial parts of H. beanii. The structure of the new compound was determined based on high-resolution electrospray ionization mass spectroscopy (HR-ESIMS), nuclear magnetic resonance (NMR), Infrared Spectroscopy (IR), ultraviolet spectrophotometry (UV) spectroscopic data. The anti-inflammatory effects of all the isolates were assessed by measuring the inhibitory effect on nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages. Compounds 3,4-dihydroxy-2-methoxyxanthone (15), 1,3,5,6-tetrahydroxyxanthone (19), and 1,3,6,7-tetrahydroxyxanthone (22) exhibited significant anti-inflammatory effects at a concentration of 10 μM with higher potency compared to the positive control quercetin. Furthermore, compounds 15, 19, and 22 reduced inducible NO synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, and cyclooxygenase 2 (COX-2) mRNA expression in the LPS-stimulated RAW 264.7 macrophages, suggesting that these compounds may mitigate the synthesis of the aforementioned molecules at the transcriptional level, provisionally confirming their anti-inflammatory efficacy.


Introduction
The genus Hypericum, a member of the Hypericaceae family, encompasses over 500 taxa distributed across temperate regions in the northern hemisphere and high-altitude tropical areas worldwide [1].In southwestern China, there are approximately fifty-five species and eight subspecies of this genus.More than 20 species from Hypericum have been utilized in traditional Chinese medicine [2].Among them, Hypericum beanii N. Robson, a perennial upright herb primarily found in temperate regions such as Kunming, Lunan, and Mengzi in Yunnan Province, China, is locally known as "Huang-hua-xiang" [3].H. beanii has been traditionally used for its therapeutic properties including heat clearing (reducing or eliminating heat-related imbalances in the body) and detoxification effects, as well as relaxation of tendons and activation of collaterals to address various ailments, such as menstrual fever, epistaxis, jaundice due to damp-heat (a pathological condition where there is an accumulation of dampness (Shi) and heat (Re) in the body), arthralgia, myalgia, upper respiratory infections, hepatitis, nephritis, and stomatitis [4,5].Recent reports have identified the presence of several phenolic compounds in H. beanii, including xanthones, flavones, and phloroglucinols.Among them, polycyclic polyprenylated acylphloroglucinols (PPAPs) have exhibited hepatoprotective, cytotoxic, and anti-inflammatory activities in vitro [6][7][8].
Inflammation is a defense response of living tissues to injurious stimuli and a complex biological response by the organism to remove the stimulus and then initiate the tissue Molecules 2024, 29, 3705 2 of 13 healing process [9].Macrophages play a crucial role in the response to inflammatory stimuli by secreting a series of pro-inflammatory cytokines, signaling proteins, and various other inflammatory mediators, including TNF-α, IL-1β, and IL-6 [10].NO regulated by iNOS reacts with peroxides to promote the inflammatory processes [11].The enzyme responsible for catalyzing the conversion of arachidonic acid into prostaglandin G2 (PGG2) and subsequently into PGH2 is known as COX, or prostaglandin-endo peroxide synthase.There are two mammalian isozymes encoded by different genes: the constitutive COX-1 and the inducible COX-2.Both enzymes are major targets of NSAIDs in pharmacology [12].
Xanthones possess a unique 9H-Xanthen-9-one scaffold, mainly found in the plants of the Gentianaceae and Hypericaceae families.Xanthones showed a variety of pharmacological activities and structural diversity.Many xanthones have been reported with potent anti-inflammatory properties [13].In order to find more xanthone compounds with anti-inflammatory activities from the aerial parts of H. beanii, in the present study, one new xanthone (1) and twenty-three known xanthone compounds  were obtained from the aerial parts of H. beanii (Figure 1).The anti-inflammatory properties of these compounds were evaluated based on their ability to inhibit NO production in LPS-stimulated RAW 264.7 macrophages.Compounds 15, 19, and 22 exhibited significant anti-inflammatory effects.Furthermore, the anti-inflammatory activity of compounds 15, 19, and 22 was confirmed through the inhibition of pro-inflammatory mediator production and other potentially associated anti-inflammatory signaling pathways.
plex biological response by the organism to remove the stimulus and then initiat sue healing process [9].Macrophages play a crucial role in the response to inflam stimuli by secreting a series of pro-inflammatory cytokines, signaling proteins, a ous other inflammatory mediators, including TNF-α, IL-1β, and IL-6 [10].NO re by iNOS reacts with peroxides to promote the inflammatory processes [11].The responsible for catalyzing the conversion of arachidonic acid into prostaglan (PGG2) and subsequently into PGH2 is known as COX, or prostaglandin-endo p synthase.There are two mammalian isozymes encoded by different genes: the con COX-1 and the inducible COX-2.Both enzymes are major targets of NSAIDs in cology [12].
Xanthones possess a unique 9H-Xanthen-9-one scaffold, mainly found in th of the Gentianaceae and Hypericaceae families.Xanthones showed a variety of cological activities and structural diversity.Many xanthones have been reported tent anti-inflammatory properties [13].In order to find more xanthone compoun anti-inflammatory activities from the aerial parts of H. beanii, in the present stu new xanthone (1) and twenty-three known xanthone compounds  were o from the aerial parts of H. beanii.(Figure 1).The anti-inflammatory properties compounds were evaluated based on their ability to inhibit NO production in LP lated RAW 264.7 macrophages.Compounds 15, 19, and 22 exhibited significant flammatory effects.Furthermore, the anti-inflammatory activity of compounds 15 22 was confirmed through the inhibition of pro-inflammatory mediator produc other potentially associated anti-inflammatory signaling pathways.

Cell Viability
The potential cytotoxicity of compounds (1-24) on RAW 264.7 macrophage cells was evaluated through Cell Counting Kit-8 (CCK-8, Apexbio, Houston, TX, USA).The results were compared to a control group incubated with normal medium only.As depicted in Figure 3, the majority of compounds exhibited no significant cytotoxic effects at a concentration of 10 µM.Consequently, we deduced that the concentration of 10 µM for further analyses would be appropriate.

Cell Viability
The potential cytotoxicity of compounds (1-24) on RAW 264.7 macrophage cells was evaluated through Cell Counting Kit-8 (CCK-8, Apexbio, Houston, TX, USA).The results were compared to a control group incubated with normal medium only.As depicted in Figure 3, the majority of compounds exhibited no significant cytotoxic effects at a concentration of 10 μM.Consequently, we deduced that the concentration of 10 μM for further analyses would be appropriate.

Evaluation of Anti-Inflammatory Activity of the Isolated Compounds
Previous reports have shown many models, either in vitro or in vivo, which are recruited to evaluate the anti-inflammatory properties of xanthones.A lot of xanthones with anti-inflammatory properties have been found, including simple oxygenated xanthones,

Evaluation of Anti-Inflammatory Activity of the Isolated Compounds
Previous reports have shown many models, either in vitro or in vivo, which are recruited to evaluate the anti-inflammatory properties of xanthones.A lot of xanthones with anti-inflammatory properties have been found, including simple oxygenated xanthones, xanthone glycosides, prenylated xanthones, and xanthonolignoids [13].Nitric oxide (NO) is a short-lived molecule produced by the enzyme nitric oxide synthase (NOS) and is released by macrophages in response to pathogens.In many diseases, high levels of NO are produced due to the induction of iNOS [37].Increased production of NO has been considered an indication of macrophage activation, making it a well-known method for investigating anti-inflammatory properties [38].
We have separated the chemical components from the aerial parts of H. beanii, in order to find more xanthone compounds with anti-inflammatory activities and to promote the reasonable use of this herb as a medicinal plant.In this study, all xanthone compounds from the aerial parts of H. beanii were tested for their inhibitory activities at 10 µM against NO production in LPS-induced RAW264.7 macrophages (Figure 4).Except for compounds 1, 11, 12, 18, 20, and 21, other components showed varying degrees of anti-inflammatory activity.Compounds 15, 19, and 22 displayed significant anti-inflammatory effects with higher potency compared to the positive control quercetin.
der to find more xanthone compounds with anti-inflammatory activities and to promote the reasonable use of this herb as a medicinal plant.In this study, all xanthone compounds from the aerial parts of H. beanii were tested for their inhibitory activities at 10 μM against NO production in LPS-induced RAW264.7 macrophages (Figure 4).Except for compounds 1, 11, 12, 18, 20, and 21, other components showed varying degrees of anti-inflammatory activity.Compounds 15, 19, and 22 displayed significant anti-inflammatory effects with higher potency compared to the positive control quercetin.
All the compounds 1-24 isolated from H. beanii were analogues and possessed the same xanthone skeleton with different amounts of -OCH3 and -OH groups at C-1 to C-7 positions.Compounds 1 and 22-24 have substituents in four identical positions of C-1, C-3, C-6, and C-7, but 1, 23, and 24 displayed much weaker anti-inflammatory activity than 22. Careful further molecular structure analysis allowed us to reach the preliminary deduction that the anti-inflammatory activity of compound was reduced when -OH was substituted by -OCH3 at the same position.Similar conclusions are observed between compounds 15 and 16, as well as between compounds 19 and 20, and 19 and 21.This result suggested that the number of phenolic hydroxyl groups is a key factor in the enhancement of the anti-inflammatory activity of xanthone when the number and position of substituents are exactly the same.Many plants of the Hypericum genus containing xanthones as bioactive constituents have been traditionally used as anti-inflammatory agents [13,39].Among the xanthone derivatives tested, compounds 15, 19, and 22 showed a remarkable inhibitory effect, suggesting its potential anti-inflammatory activity.Previous studies have highlighted the Careful further molecular structure analysis allowed us to reach the preliminary deduction that the anti-inflammatory activity of compound was reduced when -OH was substituted by -OCH 3 at the same position.Similar conclusions are observed between compounds 15 and 16, as well as between compounds 19 and 20, and 19 and 21.This result suggested that the number of phenolic hydroxyl groups is a key factor in the enhancement of the anti-inflammatory activity of xanthone when the number and position of substituents are exactly the same.

Effect of Compounds 15, 19, and 22 on NO Production and the mRNA Level of iNOS
As shown in Figure 5, the concentrations of NO and mRNA expression level of iNOS were higher in the LPS treatment group than in the control group, indicating an immune inflammatory reaction.And treatment with compounds 15, 19, and 22 resulted in a significantly lower NO content compared to that in LPS-treated cells.The expression level of iNOS mRNA, when treated with compounds 15, 19, and 22, showed a noticeable difference from the LPS group.It can be speculated that compounds 15, 19, and 22 inhibit NO synthase by suppressing iNOS expression, ultimately reducing NO production.
Many plants of the Hypericum genus containing xanthones as bioactive constituents have been traditionally used as anti-inflammatory agents [13,39].Among the xanthone derivatives tested, compounds 15, 19, and 22 showed a remarkable inhibitory effect, suggesting its potential anti-inflammatory activity.Previous studies have highlighted the ability of these compounds to modulate inflammatory pathways, including the inhibi-tion of NO production [39].In this study, the observed inhibitory effects suggest that compounds 15, 19, and 22 may interfere with the inflammatory cascade by suppressing NO production.
Molecules 2024, 29, x FOR PEER REVIEW 6 of 14 ability of these compounds to modulate inflammatory pathways, including the inhibition of NO production [39].In this study, the observed inhibitory effects suggest that compounds 15, 19, and 22 may interfere with the inflammatory cascade by suppressing NO production.

Effect of Compounds 15, 19, and 22 on TNF-α, IL-1β, and IL-6
Macrophage activation can increase the production of cytokines such as TNF-α, IL-1β, and IL-6.Overproduction and release of these cytokines can disrupt the immune balance of the body, leading to an exaggerated immune response and contributing to the development of inflammatory diseases [40].Therefore, the anti-inflammatory activity of compounds 15, 19, and 22 on TNF-α, IL-1β, and IL-6 levels was analyzed to assess their effects on the pathways involving these cytokines.
The results presented in Figure 6 show that the LPS-treated group exhibited a significant elevation in TNF-α, IL-1β, and IL-6 levels compared to the control group.However, after treatment with compounds 15, 19, and 22, concentrations of TNF-α, IL-1β, and IL-6 were reduced.We determined the effect of compounds 15, 19, and 22 on the mRNA expression of TNF-α, IL-1β, and IL-6 using qPCR.As showed in Figure 7, the mRNA expression of TNF-α, IL-1β, and IL-6 increased upon LPS treatment, following the same trend as the secretion levels of TNF-α, IL-1β, and IL-6.Conversely, following treatment with compounds 15, 19, and 22, the mRNA expression of TNF-α, IL-1β, and IL-6 in the macrophages decreased compared to the LPS-treated group, supporting our previous finding that treatment with compounds 15, 19, and 22 reduced the mRNA expression of TNF-α, IL-1β, and

Effect of Compounds 15, 19, and 22 on TNF-α, IL-1β, and IL-6
Macrophage activation can increase the production of cytokines such as TNF-α, IL-1β, and IL-6.Overproduction and release of these cytokines can disrupt the immune balance of the body, leading to an exaggerated immune response and contributing to the development of inflammatory diseases [40].Therefore, the anti-inflammatory activity of compounds 15, 19, and 22 on TNF-α, IL-1β, and IL-6 levels was analyzed to assess their effects on the pathways involving these cytokines.
The results presented in Figure 6 show that the LPS-treated group exhibited a significant elevation in TNF-α, IL-1β, and IL-6 levels compared to the control group.However, after treatment with compounds 15, 19, and 22, concentrations of TNF-α, IL-1β, and IL-6 were reduced.We determined the effect of compounds 15, 19, and 22 on the mRNA expression of TNF-α, IL-1β, and IL-6 using qPCR.As showed in Figure 7

Effect of Compounds 15, 19, and 22 on COX-2
Recent reports have suggested that increased levels of COX activity promote inflammatory pain [44].To investigate the inhibitory effect of compounds 15, 19, and 22 on LPSinduced inflammatory response in RAW 264.7 macrophages, the level of COX-2 mRNA expression was determined.After treatment with LPS, the mRNA expression levels of COX-2 were significantly increased, whereas following treatment with compounds 15, 19, and 22, the mRNA expression of COX-2 significantly decreased compared to the LPStreated group (Figure 8).Similar results were reported that suggest that ravenelin, a xanthone, suppressed iNOS and COX-2 expression in LPS-induced RAW 264.7 macrophages [45].

Effect of Compounds 15, 19, and 22 on COX-2
Recent reports have suggested that increased levels of COX activity promote inflammatory pain [44].To investigate the inhibitory effect of compounds 15, 19, and 22 on LPS-induced inflammatory response in RAW 264.7 macrophages, the level of COX-2 mRNA expression was determined.After treatment with LPS, the mRNA expression levels of COX-2 were significantly increased, whereas following treatment with compounds 15, 19, and 22, the mRNA expression of COX-2 significantly decreased compared to the LPS-treated group (Figure 8).Similar results were reported that suggest that ravenelin, a xanthone, suppressed iNOS and COX-2 expression in LPS-induced RAW 264.7 macrophages [45].

General Experimental Procedures
A Shimadzu UV-2401PC spectrophotometer was used to obtain the UV spectra.A Thermo NICOLET Is10 FT-IR spectrometer was used for IR spectroscopy with KBr pellets.The 1D and 2D NMR spectra were recorded on an Avance III-600 spectrometer with TMS as an internal standard, and chemical shifts (δ) are expressed in ppm.HR-MS was performed on an Agilent 1290 UPLC/6540 Q-TOF spectrometer (Agilent, Santa Clara, CA, USA).A JASCO J-1500 spectrometer was used to measure the ECD spectra (JASCO Corp., Japan).Sephadex LH-20 gel (25-100 μm) was obtained from Pharmacia Fine Chemical (Uppsala, Sweden), and C18 silica gel (50 μm) was procured from YMC (Osaka, Japan).Column chromatography silica gel (200-300 mesh) and thin-layer chromatography (TLC) silica gel plates were purchased from Shanghai Haohong Biomedical Technology (Shanghai, China).HPLC separations were carried out with a Thermo Scientific UltiMate3000 liquid chromatography system, and a Thermo Hypersil ODS column (ODS, 250 × 10 mm, 5 μm; Thermo, Waltham, MA, USA) was used.

Plant Material
The aerial parts of H. beanii were collected from Kunming, Yunnan Province, People's Republic of China, in June 2019 and were authenticated by Prof. Kai-Jin Wang (Anhui University).A voucher specimen (20190630-3) was deposited at the School of Pharmacy, Anhui Medical University.

General Experimental Procedures
A Shimadzu UV-2401PC spectrophotometer was used to obtain the UV spectra.A Thermo NICOLET Is10 FT-IR spectrometer was used for IR spectroscopy with KBr pellets.The 1D and 2D NMR spectra were recorded on an Avance III-600 spectrometer with TMS as an internal standard, and chemical shifts (δ) are expressed in ppm.HR-MS was performed on an Agilent 1290 UPLC/6540 Q-TOF spectrometer (Agilent, Santa Clara, CA, USA).A JASCO J-1500 spectrometer was used to measure the ECD spectra (JASCO Corp., Japan).Sephadex LH-20 gel (25-100 µm) was obtained from Pharmacia Fine Chemical (Uppsala, Sweden), and C18 silica gel (50 µm) was procured from YMC (Osaka, Japan).Column chromatography silica gel (200-300 mesh) and thin-layer chromatography (TLC) silica gel plates were purchased from Shanghai Haohong Biomedical Technology (Shanghai, China).HPLC separations were carried out with a Thermo Scientific UltiMate3000 liquid chromatography system, and a Thermo Hypersil ODS column (ODS, 250 × 10 mm, 5 µm; Thermo, Waltham, MA, USA) was used.

Plant Material
The aerial parts of H. beanii were collected from Kunming, Yunnan Province, People's Republic of China, in June 2019 and were authenticated by Prof. Kai-Jin Wang (Anhui University).A voucher specimen (20190630-3) was deposited at the School of Pharmacy, Anhui Medical University.

CCK-8 Assay
Cell viability was examined by the CCK-8 assay [46], and none of the test compounds exhibited cytotoxicity at their effective concentrations.Quercetin was used as a positive control.

Anti-Inflammatory Activity
The inhibitory effects of all the compounds on LPS-stimulated NO production were evaluated in RAW264.7 macrophages by Griess reaction [46].The cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM (Gibco, Pleasanton, CA, USA) supplemented with 10% FBS (Rongye Biotechnology, Lanzhou, China), 100 U/mL penicillin, and 100 U/mL streptomycin (Kaiji Biotechnology, Shanghai, China), and incubated at 37 • C in a humidified atmosphere with 5% CO 2 .The RAW 264.7 cells were seeded on 24-well plates at 1 × 10 5 cells/well and incubated for 24 h.Then, they were treated with compounds and quercetin (10 µM) with LPS (1 µg/mL).After 24 h, 50 µL of cell-free supernatant was mixed with 100 µL of Griess reagent and incubated at room temperature for 5 min.The concentration of nitrite was measured at 540 nm.Sodium nitrite was used as a standard to calculate the NO concentration.

Figure 4 .
Figure 4.The inhibitory effects of 1-24 against LPS-induced NO production in RAW264.7 macrophages.Mean ± SEM of three replicates is shown.* p < 0.05, ** p < 0.01, *** p < 0.001 with the LPS group.### p < 0.001 with the CON (control) group.All the compounds 1-24 isolated from H. beanii were analogues and possessed the same xanthone skeleton with different amounts of -OCH 3 and -OH groups at C-1 to C-7 positions.Compounds 1 and 22-24 have substituents in four identical positions of C-1, C-3, C-6, and C-7, but 1, 23, and 24 displayed much weaker anti-inflammatory activity than 22.Careful further molecular structure analysis allowed us to reach the preliminary deduction that the anti-inflammatory activity of compound was reduced when -OH was substituted by -OCH 3 at the same position.Similar conclusions are observed between compounds 15 and 16, as well as between compounds 19 and 20, and 19 and 21.This result suggested that the number of phenolic hydroxyl groups is a key factor in the enhancement of the anti-inflammatory activity of xanthone when the number and position of substituents are exactly the same.
The molecular formula was established as C 15 H 12 O 6 based on the quasi-molecular ion observed at m/z 287.0561 [M − H] − (calcd 287.0561 for C 15 H 11 O 6 −